🦠 Mesophilic lactic acid bacteria in food — colony-count technique on MRS agar (pH 5.7), 30 °C, 72 h
In short
1 ml portions of the sample or of the dilutions are inoculated by the pour-plate technique onto two plates, about 15 ml of MRS agar of pH 5. The test is performed according to PN-ISO 15214:2002 (ISO 15214:1998); Incubation: 30 °C ± 1 °C, 72 h ± 3 h, plates inverted; avoid the drying out of the agar (the inhibition increases). The procedure comprises 7 steps; it is used for: Fermented products: yoghurts, kefirs, cheeses, silage, fermented vegetable juices, ripened sausages — control of the starter cultures and of the number of viable bacteria, Probiotics and supplements (the number of viable Lactobacillus cells), studies of survival in a model alimentary tract, Assessment of the spoilage of products by lactic acid bacteria (vacuum-packed meat, salads, beverages).
At a glance
- Standard: PN-ISO 15214:2002 (ISO 15214:1998)
- Category: Microbiology
- Procedure steps: 7
- Incubation: 30 °C ± 1 °C, 72 h ± 3 h, plates inverted; avoid the drying out of the agar (the inhibition increases)
- Medium: MRS agar pH 5.7 ± 0.1 (25 °C); 121 °C for 15 min; about 15 ml per plate at about 47 °C
- Counting: plates with < 300 colonies at two successive dilutions, ≥ 15 colonies on at least one plate
Overview
ISO 15214:1998 specifies a horizontal method for the enumeration of viable mesophilic lactic acid bacteria by counting the colonies growing in a solid medium after incubation at 30 °C for 3 days; it is applied to products intended for human consumption and to feed. In certain products psychrotrophic or thermophilic lactic acid bacteria occur that require other temperatures, and not all lactic acid bacteria grow on MRS agar of pH 5.7 (some of them grow poorly). Mesophilic lactic acid bacteria are defined as bacteria forming colonies at 30 °C in a solid selective medium (MRS agar of pH 5.7) under the conditions of the test. Buffered peptone water does not always ensure a good resuscitation of lactic acid bacteria (annex A). In the Polish scientific literature (for example studies of the survival of Lactobacillus strains in a model alimentary tract, ŻNTJ 2015) the number of bacterial cells is determined "in accordance with PN-ISO 15214:2002 on the solid MRS medium"; in teaching (UWM) yoghurts and fermented vegetable juices are plated as 0.1 ml portions of the dilutions onto MRS plates with incubation in anaerobic containers with sachets.
Method principle
Two plates of MRS agar of pH 5.7 are prepared, inoculated by the pour-plate technique (or possibly by the spread-plate technique) with a specified amount of the liquid sample or of the initial suspension, together with successive pairs of plates from the decimal dilutions; the plates are incubated at 30 °C for 72 h; the number of bacteria per g or ml is calculated from the number of colonies on the selected plates, where appropriate after confirmation. MRS medium (de Man, Rogosa, Sharpe) per 1000 ml: casein digest 10 g, meat extract 10 g, yeast extract 4 g, triammonium citrate 2 g, sodium acetate 5 g, MgSO₄·7H₂O 0.2 g, MnSO₄·4H₂O 0.05 g, K₂HPO₄ 2 g, glucose 20 g, Tween 80 1.08 g, agar 12–18 g; the components are dissolved by boiling, the pH is adjusted so that after sterilisation it is 5.7 ± 0.1 at 25 °C (a tolerance of ± 0.1, so that the pH does not fall below 5.6), it is sterilised for 15 min at 121 °C and cooled to about 47 °C (stored medium is melted in a boiling bath and cooled to 47 °C). Where there is a risk of heavy contamination with yeasts (e.g. dried sausages) sorbic acid is added: 1.4 g dissolved in about 10 ml of 1 mol/l NaOH, sterilised by filtration, to 1000 ml of MRS cooled to 47 °C, the final pH being 5.7 ± 0.1. Ready-made commercial media are allowed, but their composition and pH may differ and give different results. The spread-plate technique with anaerobic or microaerobic incubation (candle jars) and a double-layer medium are allowed. Some Leuconostoc form large slimy colonies that hinder the development of the others (an underestimate); because other micro-organisms may grow on MRS in certain products the colonies are confirmed by simple tests (Gram staining, catalase) — this being noted in the test report. The calculation: N = ΣC / [V·(n₁ + 0.1·n₂)·d], rounded to two significant figures; where there are < 15 colonies on the plates from the sample/initial suspension an estimated number is reported (the arithmetic mean y; NE = y for liquid products, y/d for the others).
Applications
- Fermented products: yoghurts, kefirs, cheeses, silage, fermented vegetable juices, ripened sausages — control of the starter cultures and of the number of viable bacteria
- Probiotics and supplements (the number of viable Lactobacillus cells), studies of survival in a model alimentary tract
- Assessment of the spoilage of products by lactic acid bacteria (vacuum-packed meat, salads, beverages)
- Feed and silage (inoculants), plant raw materials
- Quality control of the MRS medium and of the cultures in dairying
Key parameters
| Parameter | Value |
|---|---|
| Incubation | 30 °C ± 1 °C, 72 h ± 3 h, plates inverted; avoid the drying out of the agar (the inhibition increases) |
| Medium | MRS agar pH 5.7 ± 0.1 (25 °C); 121 °C for 15 min; about 15 ml per plate at about 47 °C |
| Counting | plates with < 300 colonies at two successive dilutions, ≥ 15 colonies on at least one plate |
| Result | N = ΣC/[V(n₁ + 0.1n₂)d], 2 significant figures, as 1.0–9.9 × 10ⁿ per g or ml; an estimated number where there are < 15 colonies |
| Additive against yeasts | sorbic acid 1.4 g/l (in 10 ml of 1 mol/l NaOH, filtered) |
| Variants | spread plate + anaerobic/microaerobic incubation (candle jars); double-layer MRS |
| Confirmation (optional) | Gram staining, catalase — for products in which other micro-organisms grow on MRS |
Standard
- Standard number
- PN-ISO 15214:2002 (ISO 15214:1998)
- Title (PL)
- Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby mezofilnych bakterii fermentacji mlekowej — Metoda płytkowa w temperaturze 30 stopni C
- Title (EN)
- Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of mesophilic lactic acid bacteria — Colony-count technique at 30 °C
Step-by-step procedure
-
Medium
Dissolve the components of MRS by boiling, adjust the pH so that after sterilisation it is 5.7 ± 0.1 at 25 °C, sterilise for 15 min at 121 °C and cool to about 47 °C (melt stored medium in a boiling bath and cool it to 47 °C); where there is a risk of yeasts add the filtered solution of sorbic acid (1.4 g in about 10 ml of 1 mol/l NaOH per 1 l).
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Sample and dilutions
Prepare the initial suspension and the decimal dilutions in accordance with ISO 6887-1; the sample is to be representative and undamaged in transport.
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Inoculation
Transfer 1 ml portions of the liquid sample or of the initial suspension into two sterile plates, and 1 ml portions of the successive dilutions into the further pairs (a new pipette for each dilution); where the numbers are high plate only the dilutions needed for the counting.
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Pouring
Pour about 15 ml of MRS at a temperature of about 47 °C into each plate, mix it carefully and leave it to set (alternatively the spread-plate technique with anaerobic or microaerobic incubation, or double-layer MRS).
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Incubation
Incubate the inverted plates at 30 °C for 72 h ± 3 h, protecting the agar from drying out.
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Counting and confirmation
Count the colonies on the plates with < 300 colonies at two successive dilutions and with ≥ 15 on at least one of them; take into account the possible slimy colonies of Leuconostoc; where necessary confirm the colonies by Gram staining and the catalase test (note this in the test report).
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Calculation
N = ΣC/[V(n₁ + 0.1n₂)d] (e.g. (168 + 215 + 14 + 25)/[1·(2 + 0.2)·10⁻²] = 19 182 → 1.9 × 10⁴/g); where there are < 15 colonies — the estimated number NE = y (liquid) or y/d (others); the result as 1.0–9.9 × 10ⁿ per g or ml.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator 30 °C ± 1 °C | — | |
| Water bath 47 °C ± 2 °C and boiling bath | cooling and melting of the medium | — |
| Petri dishes of 90–100 mm, pipettes of 10 ml (0.5 ml) and 1 ml (0.1 ml) | — | |
| pH meter (0.01; ± 0.1 pH at 25 °C) | adjustment of the pH to 5.7 | — |
| Autoclave and drying oven; optionally anaerobic/candle jars | in accordance with ISO 7218 | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| MRS agar (casein digest 10 g, meat extract 10 g, yeast extract 4 g, triammonium citrate 2 g, sodium acetate 5 g, MgSO₄·7H₂O 0.2 g, MnSO₄·4H₂O 0.05 g, K₂HPO₄ 2 g, glucose 20 g, Tween 80 1.08 g, agar 12–18 g/l), pH 5.7 | — | solid selective medium for lactic acid bacteria |
| Sorbic acid 1.4 g + NaOH 1 mol/l (about 10 ml), sterilised by filtration | — | inhibition of yeasts (dried products, sausages) |
| Diluents in accordance with ISO 6887-1 | — | initial suspension and dilutions (buffered peptone water does not always resuscitate LAB) |
| Reagents for Gram staining, H₂O₂ for the catalase test | — | optional confirmation of the colonies |
Health and safety (OHS)
- Lactic acid bacteria are not pathogenic, but food samples may contain pathogens — good microbiological practice
- Hot agar (47 °C) and the boiling bath — protection against burns; 1 mol/l NaOH — caustic
- Anaerobic jars with a candle or with generators — care with naked flames and with hydrogen
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-ISO 15214:2002 (ISO 15214:1998) — “Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of mesophilic lactic acid bacteria — Colony-count technique at 30 °C”.
How does this method work?
Two plates of MRS agar of pH 5.7 are prepared, inoculated by the pour-plate technique (or possibly by the spread-plate technique) with a specified amount of the liquid sample or of the initial suspension, together with successive pairs of plates from the decimal dilutions; the plates are incubated at 30 °C for 72 h; the number of bacteria per g or ml is calculated from the number of colonies on the selected plates, where appropriate after confirmation.
What is the measuring range and accuracy?
Incubation: 30 °C ± 1 °C, 72 h ± 3 h, plates inverted; avoid the drying out of the agar (the inhibition increases); Medium: MRS agar pH 5.7 ± 0.1 (25 °C); 121 °C for 15 min; about 15 ml per plate at about 47 °C; Counting: plates with < 300 colonies at two successive dilutions, ≥ 15 colonies on at least one plate; Result: N = ΣC/[V(n₁ + 0.1n₂)d], 2 significant figures, as 1.0–9.9 × 10ⁿ per g or ml; an estimated number where there are < 15 colonies.
How long does the test take?
The procedure comprises 7 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Incubator 30 °C ± 1 °C, Water bath 47 °C ± 2 °C and boiling bath, Petri dishes of 90–100 mm, pipettes of 10 ml (0.5 ml) and 1 ml (0.1 ml), pH meter (0.01; ± 0.1 pH at 25 °C), Autoclave and drying oven; optionally anaerobic/candle jars.
Where is this test used?
Fermented products: yoghurts, kefirs, cheeses, silage, fermented vegetable juices, ripened sausages — control of the starter cultures and of the number of viable bacteria; Probiotics and supplements (the number of viable Lactobacillus cells), studies of survival in a model alimentary tract; Assessment of the spoilage of products by lactic acid bacteria (vacuum-packed meat, salads, beverages); Feed and silage (inoculants), plant raw materials; Quality control of the MRS medium and of the cultures in dairying.
What safety precautions apply?
Lactic acid bacteria are not pathogenic, but food samples may contain pathogens — good microbiological practice; Hot agar (47 °C) and the boiling bath — protection against burns; 1 mol/l NaOH — caustic; Anaerobic jars with a candle or with generators — care with naked flames and with hydrogen.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-ISO 15214.